Intrinsic regulations in neural fate commitment - Wiley Online Library

5 downloads 565 Views 274KB Size Report
Intrinsic regulations in neural fate commitment. Ke Tang,1* Guangdun Peng,2 Yunbo Qiao,2 Lu Song2 and Naihe Jing2*. 1Institute of Life Science, Nanchang ...
The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2015) 57, 109–120

doi: 10.1111/dgd.12204

Review Article

Intrinsic regulations in neural fate commitment Ke Tang, 1 * Guangdun Peng, 2 Yunbo Qiao, 2 Lu Song 2 and Naihe Jing 2 * 1

Institute of Life Science, Nanchang University, Nanchang, Jiangxi 330031, 2State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

Neural fate commitment is an early embryonic event that a group of cells in ectoderm, which do not ingress through primitive streak, acquire a neural fate but not epidermal or mesodermal lineages. Several extracellular signaling pathways initiated by the secreted proteins bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), wingless/int class proteins (WNTs) and Nodal play essential roles in the specification of the neural plate. Accumulating evidence from the studies on mouse and pluripotent embryonic stem cells reveals that except for the extracellular signals, the intracellular molecules, including both transcriptional and epigenetic factors, participate in the modulation of neural fate commitment as well. In the review, we mainly focus on recent findings that the initiation of the nervous system is elaborately regulated by the intrinsic programs, which are mediated by transcriptional factors such as Sox2, Zfp521, Sip1 and Pou3f1, as well as epigenetic modifications, including histone methylation/demethylation, histone acetylation/deacetylation, and DNA methylation/ demethylation. The discovery of the intrinsic regulatory machineries provides better understanding of the mechanisms by which the neural fate commitment is ensured by the cooperation between extracellular factors and intracellular molecules. Key words: embryonic stem cell, epigenetic modification, extracellular signals, neural fate commitment, transcription factor.

Introduction Neural fate commitment is an early embryonic event that a group of cells in ectoderm, which do not ingress through primitive streak, acquire a neural fate but not epidermal or mesodermal lineages. The initial observations in amphibian revealed that transplanted dorsal lip of blastopore could induce a second axis at the ventral part of recipient embryo at the gastrulation stage, suggesting that the instructive interaction between dorsal lip of blastopore and surrounding ectoderm leads to the generation of the nervous system (Spemann 1921; Spemann & Mangold 1924). Evidence from different species supports that in addition to the dorsal lip of amphibian blastopore, the shield of teleosts, the Hensen’s node in birds and mammals could also participate in the induction of the neural plate in

*Author to whom all correspondence should be addressed. Emails: [email protected] and [email protected] Received 29 October 2014; revised 11 December 2014; accepted 21 December 2014. ª 2015 Japanese Society of Developmental Biologists

transplantation experiments performed in the same species, as well as in the different species, which indicates that mechanisms involved in the neural fate commitment are evolutionarily conserved in all vertebrates (Waddington 1932, 1933, 1934, 1936; Oppenheimer 1936a,b; Kintner & Dodd 1991; Blum et al. 1992; Hatta & Takahashi 1996). Many laboratories had worked hard to identify the factors that ensure the neural fate for decades. Until 20 years ago, the first molecular explanation for the initiation of neural tissue was proposed that during gastrulation, ectoderm cells will autonomously differentiate into neural fate, which is inhibited by bone morphogenetic proteins (BMPs) , especially BMP4 (Hemmatibrivanlou & Melton 1997; Munoz-Sanjuan & Brivanlou 2002; De Robertis & Kuroda 2004; Stern 2005, 2006; Vonica & Brivanlou 2006). Nevertheless, the problems in the initiation of nervous system can’t be fully solved by BMP signaling alone. Accumulating evidence from different organisms reveals that other secreted proteins, such as fibroblast growth factors (FGFs), wingless/int class proteins (WNTs) and Nodal, are also responsible for the neural fate commitment. The contribution of these classic extracellular

110

K. Tang et al.

regulations on neural induction has been well reviewed (Sasai & Derobertis 1997; Wilson & Edlund 2001; Munoz-Sanjuan & Brivanlou 2002; Stern 2005, 2006). Recent studies in mouse and pluripotent embryonic stem cells (ESCs) shed new light on how extracellular signaling pathways, especially BMP and FGF pathways, orchestrate with each other to modulate the neural lineage commitment. Loss-of-function of Bmpr1a, which is the only type I BMP receptor expressed in the epiblast of implanted mouse embryos (Mishina et al. 1995), results in premature neural differentiation of the epiblast (Di-Gregorio et al. 2007). BMP signaling pathway plays similar functions in neural differentiation of mouse ESCs in vitro (Finley et al. 1999; Tropepe et al. 2001). One of our recent studies in mouse ESCs and epiblast stem cells (EpiSCs) revealed that BMP4 plays distinct roles at different stages during the ESCs neural differentiation. BMP4 limits the generation of ESC-derived EpiSCs (ESD-EpiSCs) through repression of FGF-Erk signaling in ESCs. BMP4 suppresses the transition from ESDEpiSCs to neural progenitor cells (NPCs), and enhances the differentiation of non-neural lineages, which is probably achieved partially through the upregulation of Id genes (Zhang et al., 2010). Clearly, the BMP inhibition in neural induction is conserved in mouse. The lethality of Fgf4 / or Fgfr2 / null mouse in peri-implantation prevents to investigate their function in early neural development (Feldman et al. 1995; Arman et al. 1998). The axis formation, mesoderm specification, neuroectoderm patterning are compromised with the expanded expression of anterior neuroectoderm markers in Fgf8 / mutants, which die at late gastrulation (Meyers et al. 1998; Sun et al. 1999), indicating that FGF signaling may negatively regulate the neural lineage specification in the early mouse embryos in vivo. In mESCs in vitro, the inhibition of FGF/Erk leads to the impaired neural fate commitment (Stavridis et al. 2007). After withdrawal of Leukemia inhibitory factor (LIF), Fgf4 / mESCs resist neural differentiation, as well as differentiation in response to BMP4; instead, Fgf4 / cells persist in an undifferentiated state, retaining the expression of the pluripotency marker Oct4 and the neural differentiation capacity (Kunath et al. 2007). When FGF4 is added in the culture system, Fgf4 / mESCs regain the capability for both the neural differentiation and the differentiation response to BMP. Erk2 / ESCs phenocopy those of Fgf4 / ESCs, indicating that an intact FGF signaling pathway is imperative for the switch of BMP signaling from maintaining self-renewal to promoting non-neural differentiation (Kunath et al. 2007). In turn, FGF signaling does not actively induce neural fate commitment; in contrast, it promotes ESCs to exit na€ıve state and ª 2015 Japanese Society of Developmental Biologists

to enter a status, in which they are more ready for neural differentiation with the influence of other signals. In addition, FGF signaling is crucial for the transition from ESCs to the epiblast (Sterneckert et al. 2010), as well as the maintenance of EpiSC pluripotency (Brons et al. 2007; Tesar et al. 2007). Except for the extrinsic pathways, accumulating studies in vivo and in vitro uncover that intrinsic networks, programmed by both transcriptional and epigenetic factors, participate in the regulation of the neural fate commitment as well. Here, we are mainly describing some new findings for the old question, how the initiation of the neural plate is achieved. We apologize that because of limited space, we are not able to include all the findings about the neural fate initiation in the article.

Transcriptional factors and neural fate commitment Sox2, the first general marker of the neural plate, and beyond Sox2 gene, encoding an Sry-related HMG box transcription factor (Gubbay et al. 1990), is considered to be the first general marker of the initiating neural tissue. During the early mouse development, the expression of Sox2 transcripts is detected first in some cells at E2.5 morula, in cells within inner cell mass (ICM) at E3.5, and the whole epiblast at E6.5. During gastrulation, the expression of Sox2 is restricted to the anterior ectoderm, where there is presumptive neuroectoderm at E7.0–E8.0. The transcripts of Sox2 are examined in the headfolds and the neural tube at E8.5, and throughout the brain and the neural tuble at E9.5. (Rex et al. 1997; Wood & Episkopou 1999; Avilion et al. 2003; Bylund et al. 2003; Graham et al. 2003; Uchikawa et al. 2003; Tanaka et al. 2004). In vitro, Sox2 is expressed in the original ESCs, and during the earliest stages of ESC neural differentiation (Thomson et al. 2011). The expression profiles of Sox2 gene suggest that it may play an essential role in the early neural development. Sox2 homozygous mutants are lethal at peri-implantation stage. Blastocyst injection assays reveal that Sox2 plays a cell-autonomous function in the development of epiblast cells (Avilion et al. 2003). At the Sox2 genomic locus, more than 23 enhancers have been identified. N1 and N2 enhancers are crucial to the process of the neural fate determination. In the early mouse embryo, the N2 enhancer mediates the expression of Sox2 in the epiblast and the anterior neural plate (Iwafuchi-Doi et al. 2011). In chick embryos, the activation of the N1 enhancer is relatively late (Uchika-

Intrinsic regulations in neural fate commitment

wa et al. 2003). Tbx6 represses the N1 enhancer activity in cells acquiring paraxial mesodermal fate. The persisting activation of N1 enhancer turns paraxial mesoderm into neural lineages (Takemoto et al. 2011). In mESCs, Sox2 and other transcription factors such as Oct4, Nanog generate complicated regulatory networks to maintain the pluripotent state (Boyer et al. 2005; Masui et al. 2007; Chen et al. 2008; Kim et al. 2008; Orkin & Hochedlinger 2011). During the differentiation of mESCs, the expression of Oct4 is increased in cells acquiring the mesendoderm fate, but decreased in cells acquiring the neuroectoderm identity. In contrast, the expression of Sox2 is enhanced in cells gaining the neural fate and reduced in cells gaining the mesendoderm fate. Sox2 might preferentially promote neuroectoderm differentiation by blocking the key regulators such as Brachyury in the development of mesendoderm lineages (Thomson et al. 2011; Wang et al. 2012). Clearly, Sox2 is not only the first general marker of the neural tissue, but also an imperative regulator ensuring the neural fate. Zfp521 is a crucial neural initiating nuclear factor Zfp521 gene, encoding a zinc-finger nuclear protein (Warming et al. 2003; Bond et al. 2008), is exclusively expressed in the anterior neuroectoderm of mouse embryo during the gastrulation at E7.5 and in the neuroectoderm of the rostral neural tube after the gastrulation at E8.0 (Kamiya et al. 2011). Zfp521 is not expressed in the original ESCs, and its expression is activated dramatically during the neural differentiation of ESCs in serum-free culture of embryoid-bodylike aggregates. The expression profiles of Zfp521 suggest that it may play a critical role in the initiation of the neural fate. Forced expression of Zfp521 enhances the neural conversion of ESCs, and its neural promoting activity could even override the neural inhibitory effects of BMP4. In contrast, the neural differentiation of Zfp521 deficient mESCs is compromised. Co-culture assays with the Zfp521-shRNA ESCs and the control ESCs demonstrate that Zfp521 may regulate the neural differentiation in a cell-autonomous manner. Clearly, Zfp521 is required and sufficient for the neural conversion of ESCs (Kamiya et al. 2011). Epiblast-like cells but not neuroectodermal cells could be generated in the differentiated Zfp521 depleting ESCs, suggesting that Zfp521 plays a specific function to drive the transition from epiblast to neuroectoderm. Chromatin immunoprecipitation (ChIP) assay reveals that Zfp521 protein is specifically enriched at the genomic regions of the neuroectoderm-specific genes such as Sox1, Sox3 and Pax6. Zfp521 may

111

coordinate with co-activator p300 to directly activate these early neuroectodermal genes. Thus, Zfp521 specifically initiates the intrinsic transition of mESCs differentiation into NPCs (Kamiya et al. 2011). SIP1 is essential for the neural fate initiation Sip1, Smad-interacting protein 1, is one of the ZFHX1 family members, and possesses a homeodomain-like motif, two zinc finger clusters, and a Smad-interacting domain (Verschueren et al. 1999; Postigo et al. 2003). Sip1 can function as a repressor to inhibit the expression of genes such as E-cadherin and Brachyury through binding onto CACCT(G) site (Lerchner et al. 2000; Comijn et al. 2001). Sip1 can also interact with the activated Smad1/2/3 proteins to block BMP and ACTIVIN-NODAL signaling pathways. As a major target of Churchill, Sip1 mediates the formation of the neural plate in early chicken embryos (Sheng et al. 2003). Interestingly, several conserved Sip1 bindings sites are identified in Sox2 gene enhancers, indicating that Sip1 is a possible upstream regulator of Sox2 gene (Uchikawa et al. 2003). The expression of SIP1 is detected in pluripotent human embryonic stem cells (hESCs), and increased gradually following the neural differentiation. SIP1 expression is sharply upregulated on the inhibition of ACTIVIN-NODAL signaling by SB431542 (Chng et al. 2010). Overexpression of SIP1 reduces the expression of the pluripotency marker NANOG in hESCs, but increases the expression of both early (GBX2 and HOXA1) and later (OLIG3, SOX1 and SIX1) neural markers. In addition, SIP1 overexpression enhances the neural differentiation of both hESCs and mEpiSCs, suggesting that the neural promoting function of SIP1 is evolutionarily conserved (Chng et al. 2010). In contrast, SIP1 depletion increases the expression of NANOG in hESCs, and decreases the basal levels of early (GBX2, HOXA1) and later (OLIG3, SOX1) neural genes. Interestingly, during the neural differentiation of hESCs, it is the expression of later neural markers OLIO3, SOX1 and SIX1 that is reduced dramatically, indicating that SIP1 is required for the late neural differentiation (Chng et al. 2010). SIP1 may ensure the neural fate by blocking signaling pathways, which promote the progress of other germ layers, especially mesendoderm and definitive endoderm (Chng et al. 2010). Pou3f1, a dual regulator in the neural fate commitment Pou3f1, also known as Oct6, Tst1, or SCIP, encodes a POU domain transcription factor, and belongs to POU III subfamily (Monuki et al. 1989; Xi et al. 1989; ª 2015 Japanese Society of Developmental Biologists

112

K. Tang et al.

Meijer et al. 1990; Suzuki et al. 1990). The expression of Pou3f1 is detected throughout the epiblast in mouse embryos at E5.5. At E6.5 and E7.0, Pou3f1 expression was limited to the anterior part of the embryos, where the neuroectoderm is originated. The expression of Pou3f1 is restricted at the anterior neuroectoderm at E7.5 and E8.0, and is readily detected at the forebrain and the midbrain at E9.5 (Xi et al. 1989; Zwart et al. 1996; Iwafuchi-Doi et al. 2012; Zhu et al. 2014). In cell culture in vitro, Pou3f1 gene is expressed in undifferentiated ESCs (Scholer et al. 1989; Meijer et al. 1990; Zhu et al. 2014). The expression of Pou3f1 is significantly enhanced during the neural differentiation of pluripotent ESCs (Zhu et al. 2014). In addition, RA-treatment quickly activates Pou3f1 expression in P19 cell aggregates (Meijer et al. 1990; Zhu et al. 2014). The expression of Pou3f1 is high in undifferentiated EpiSCs, and is gradually reduced at day 1 and day 2 in the NPC condition (Iwafuchi-Doi et al. 2012). The expression patterns of Pou3f1 indicate that it may play an important role during the early neural development. The neural fate commitment is compromised in the neural differentiation of Pou3f1 deficient ESCs. In contrast, overexpression of Pou3f1 promotes the neural differentiation of ESCs, especially the neural transition from epiblast to neural progenitor cells, in a cell autonomous manner. The blastocyst injection assays reveal further that Pou3f1-deficient ESCs fail to integrate into the neuroectoderm in the chimeric embryos, whereas Pou3f1-overexpressing ESCs preferentially incorporate into the neuroectoderm. Thus, Pou3f1 is necessary and sufficient for the neural fate commitment of pluripotent stem cells in vivo and in vitro (Zhu et al. 2014). Genome-wide ChIP-seq and RNA-seq assays reveal that Pou3f1 might bind and activate the expression of neural-related genes, such as Sox2, Zfp521, Zic1, Zic2. Pou3f1 is enriched at the N2 enhancer region of Sox2 gene to promote the expression of the gene (Zhu et al. 2014). Consistently, the forced expression of Pou3f1 in EpiSCs also leads to the increased expression of several neural genes, including Sox1, Sip1, Otx1, Hesx1, Pax6 and Gbx2 (Iwafuchi-Doi et al. 2012). In serum-free culture of embryoid-body like aggregates, the expression of Pou3f1 is not altered by either overexpression or depletion of Zfp521. Nevertheless, the forced expression of Pou3f1 in ESCs enhances the expression of Zfp521 and early neuroectodermal marker genes in neural differentiation, which doesn’t occur in the presence of BMP4 or in Zfp521depleted ESCs. Clearly, the neuralizing activity of Pou3f1 depends on Zfp521 (Kamiya et al. 2011). In addition, Pou3f1 inhibits BMP and WNT signaling pathways by alleviating the transcription of their downª 2015 Japanese Society of Developmental Biologists

stream effector genes, such as Id1, Id2, Msx1, Msx2 of BMP pathway and Wnt3, Axin2, Dkk1, Myc of WNT pathway. Thus, Pou3f1 gene plays dual functions to enhance the neural differentiation of pluripotent stem cells, through activating intrinsic neural promoting networks, and blocking extrinsic neural inhibitory signals (Zhu et al. 2014) (Fig. 1). Transcription factor regulatory networks in the neural fate commitment Sox2, Zfp521, Sip1, and Pou3f1, as well as many other transcription factors create regulatory networks to ensure the neural fate commitment. In the process from EpiESCs to NPCs, Zic2/3, Otx2, Sox2 and Pou5f1 factors may maintain the pluripotent state of the epiblast by supporting the expression of Fgf5 and Eomes. Zic2/3, Otx2 and Pou5f1/3f1 factors block the development of mesodermal lineages by repressing T. Zic2/3 and Sox2 factors inhibit endodermal fate commitment by repressing Sox17 and Eomes. Otx2 suppresses the formation of posterior neural plate

Fig. 1. Model of Pou3f1 gene function in the neural fate commitment. During gastrulation in the early mouse embryo, Pou3f1 promotes the neural fate commitment through dual actions, activating the neural lineage genes including Zfp521, Sox2, and Zic2 in the anterior ectoderm and inhibiting the external signaling pathways such as bone morphogenetic protein (BMP) signal from the extra-embryonic areas and wingless/int class protein (WNT) signal from the posterior embryo.

Intrinsic regulations in neural fate commitment

development by repressing Nkx1.2, Gbx2 and Sox1 (Iwafuchi-Doi et al. 2012). In addition, Sox2, as well as Sox1 and Sox3, may cooperate with each other to direct the neural fate initiation during the early mouse embryo development (Graham et al. 2003). In hESCs, SMAD2, NANOG, and OCT4 inhibit SIP1 expression, while SOX2 promote the expression of SIP1 (Chng et al. 2010). In the neural differentiation of mESCs, Pou3f1 functions as an upstream regulator of Sox2 and Zfp521 (Kamiya et al. 2011; Zhu et al. 2014). Brn2, another POU III transcription factor, may partially compensate the Pou3f1 depletion (Zhu et al. 2014). Clearly, during the initiation of the neural tissue, multiple transcription factors orchestrate with each other to activate the expression of neural ectoderm genes, but to inhibit the expression of the pluripotent gene and the non-neural lineage genes. The generation of in vivo or in vitro models with the genome editing on multiple genes will broaden our understanding of mechanisms involved in the neural fate commitment.

Epigenetic regulations and neural fate commitment The studies on pluripotent embryonic stem cells in vivo and in vitro demonstrate that epigenetic regulation also play imperative roles in the neural fate commitment. The epigenetic modifications on histones or DNA control the state of chromatin, which determine whether DNA is accessible to transcription factors or not. In the process from ESCs to the neural lineages, pluripotency genes become inaccessible to transcription factors, and their expression is switched off. In contrast, neural development genes become accessible to transcription factors, and their expression is switched on, which in turn endows cells a neural fate. The dynamic changes of chromatin conformation are achieved by histone methylations such as H3K4me3, H3K27m3, H3k9me3, DNA methylation/demethylation, and histone acetylation/deacetylation (Hsieh et al. 2004; Hsieh & Gage 2005; Hirabayashi & Gotoh 2010; Coskun et al. 2012; Roidl & Hacker 2014). The bivalent state in pluripotent ESCs The pluripotent ESCs remain in an undifferentiated state. In ESCs, the expression of key regulatory genes related to the lineage commitment are silenced or maintained at low levels. Different from the permanent silencing in the differentiated cells, the silencing of these genes in ESCs is reversible, and can be alleviated by the appropriate development cues. Histone lysine methylation regulates gene transcription as well as replication and recombination (Zhang & Reinberg

113

2001). The lysine residues of histone H3 and H4 can be mono-, di-, and tri-methylated. H3K27me3 and H3K4me3 are associated with the gene repression and the gene activation, respectively. The combination of H3K27me3 and H3K4me3 is considered a mark for the bivalent chromatin, the genes in which are poised for transcriptional activation upon the differentiation of the pluripotent ESCs (Azuara et al. 2006; Bernstein et al. 2006). H3K27 methylation H3K27me3 is enriched on key developmental genes that are silenced in the pluripotent ESCs (Boyer et al. 2006; Bracken et al. 2006; Lee et al. 2006). During the transition from ESCs to NPCs, H3K27me3 is lost at the promoters of many neural lineage genes, which leads to the gene activation (Bernstein et al. 2006; Dahl et al. 2007; Mikkelsen et al. 2007; Pasini et al. 2007). H3K27 methylation is conducted by either KMT6A or KMT6B (Cao & Zhang 2004). As an H3K27 specific methyltransferase, KMT6A plays essential function for the renewal, maintenance and differentiation of ESCs (Lee et al. 2007; Sher et al. 2008). mESCs are maintained in an undifferentiated status with the presence of the cytokine leukemia inhibitor factor (LIF). After the withdrawal of LIF, retinoic acid (RA) could induce the neural differentiation of mESCs (Ross et al. 2000; Glaser & Brustle 2005). High levels of H3K27me3 and PRC2, including KMT6A, binding are detected in undifferentiated ESCs. A global decrease in H3K27me3 and loss of both KMT6A protein and mRNA are observed in differentiated ESCs after 3 days of RA treatment, which is associated with the regulation of Nestin expression. In contrast, a dynamic, rapid loss of H3K27me3 and KMT6A binding after only a few hours of RA treatment is assessed at RA direct response genes, such as Hoxa1 (Lee et al. 2007). Further study reveals that loss of the H3K27me3 repressive mark and other multiple other histone modifications, including H3K4me3 and acetylation on histones H3 and H4, are required to activate Hoxa1 (Lee et al. 2007). KDM6A, KDM6B, KDM7A and KDM7C are involved in the demethylation of H3K27me3. Compared with other histone lysine demethylase genes, Kdm6b shows the greatest upregulation at day 8 with a decrease to near basal levels at day 26 in neural differentiation of mESCs (Burgold et al. 2008), indicating that Kdm6b could be involved in the onset of neural commitment. Indeed, shRNAs mediated knockdown of Kdm6b leads to the compromised transition from ESCs to NPCs in neural differentiation, accompanying with failed upregulation of neural marker genes including Pax6, Nestin and Sox1. ChIPª 2015 Japanese Society of Developmental Biologists

114

K. Tang et al.

qPCR assays demonstrate that Pax6, Nestin and Sox1 are direct targets of KDM6B. Even though KDM6B targets show distinct patterns of H3K27 methylation and expression profiles, KDM6B is required for the neural fate commitment by regulating their expression (Burgold et al. 2008). H3K4 methylation H3K4 methylations (me-, di-, and tri-methylation) are often present at the promoter region or the transcription start site of genes transcribed, and are essential to recruit RNA polymerase and transcription factors for the gene activation. H3K4me3 demethylation leads to the transcriptional repression of target genes (Sims et al. 2003; Szutorisz et al. 2005). Methylation of H3K4 is performed by KMT2A, KMT2B, KMT2C, KMT2D, KMT2E, KMT2F, KMT2G, KMT2H, KMT3D, KMT3E and KMT7. H3K4 demethylation is catalyzed by KDM1A, KDM1B, KDM5A, KDM5B, KDM5C and KDM5D. KDM5B, a specific demethylase of methylated histone H3 lysine 4, is highly expressed in mouse epiblast at E5.5 (Frankenberg et al. 2007) and regulates G0-to-G1 progression through cell cycle checkpoint genes (Yamane et al. 2007). Transient overexpression of Kdm5b results in dramatic reduction of H3K4me3 in mESCs. The expression of Egr1, BMI1, and p27 genes, involved in cell cycle control and cell lineage commitment, is also significantly decreased. The enrichment of H3K4me3 at their promoter is reduced; in contrast, the recruitment of KDM5B is enhanced, indicating Egr1, BMI1, and p27 are direct targets of KDM5B. Constitutive Kdm5b overexpressing ESCs (mESCsKDM5b) shows a similar percentage of Oct4-positive cells as the control ESCs, but with a higher rate of mitosis. mESCKDM5b can differentiate to neurospheres (NSKDM5b). But compared with the control, the proliferation of NSKDM5b increases two folds, and in the replating process, NSKDM5b continues to grow in suspension. The expression of Nanog is also significant higher in mESCsKDM5b, probably due to the repressed expression of Tcf3, which inhibits Nanog expression. Clearly, KDM5B may inhibit the expression of regulatory genes such as Tcf3, BMI1, Egr1, and p27 to modulate the proliferation and differentiation of ESCs (Dey et al. 2008). In addition, KDM5B is mainly localized at transcription start sites of development-related genes to mediate the transition from ESCs to NPCs (Schmitz et al. 2011). Interestingly, transcriptional regulation by KDM5C depends on the loci of genomic element bound. The KDM5C demethylase protein is detected in mouse ESCs and NPCs. ChIP-seq assays of KDM5C, H3K4me1 or H3K4me3 reveal that KDM5C peaks overlaps within ª 2015 Japanese Society of Developmental Biologists

regions of high, intermediate, or low levels of H3K4me3. High H3K4me3 regions close to a TSS correspond to active promoters, with high signals for H3K27ac and for global run-on sequencing (GROseq). In contrast, regions with intermediate or low H3K4me3 levels are positive for H3K4me1 with lower but significant H3K27ac or GRO-seq signals (Outchkourov et al. 2013). Kdm5c knockdown results in localized increases in H3K4me3 and reductions in H3K4me1 modifications, but not the global H3K4me3 levels. Genes associated with KDM5C binding sites in high H3K4me3 promoters are mainly upregulated. Nevertheless, genes containing Kdm5c peaks with intermediate- and low-H3K4me3 enhancer elements are mainly downregulated. Promoter and enhancer activity assays are performed in ESCs with two luciferase reporters, pGL3-basic for promoter activity and pPou5F1 for enhancer activity. Luciferase data confirmed it further that KDM5C inhibits gene transcription at promoters, but stimulates gene expression at enhancers by balancing of Histone H3K4 methylation states (Outchkourov et al. 2013). During the transition from mESCs to NPCs, the binding of KDM5C at enhancer regions is lost, indicating that Kdm5c may participate in the regulation of neural fate commitment (Outchkourov et al. 2013). The detailed underlining mechanisms still need to be clarified. H3K9 methylation As H3K27me3, H3K9me3 is also a mark for gene inhibition. Methylation of H3K9 is performed by KMT1A, KMT1B, KMT1C, KMT1D, KMT1E, KMT1F, and KMT8A. H3K9 demethylation is catalyzed by KDM1A, KDM1B, KDM3A, KDM3B, KDM4A, KDM4B, KDM4C, KDM4D, KDM7A, KDM7B and KDM7C. H3K9 is not related to the bivalent state of ESCs. Di- and trimethylations of H3K9 are observed in the pluripotent genes and non-neural lineage genes during the differentiation between hESCs to NPCs, indicating that H3K9 methylation leads to the silencing of these genes (Golebiewska et al. 2009). KDM4C can demethylate H3K9me3 to activate gene expression (Wissmann et al. 2007). The expression of Kdm7a is increased at the early stage of mESC neural differentiation. The process of ESCs to NPCs is compromised in Kdm7a deficient ESCs, whereas overexpression of Kdm7a promotes the neural differentiation. In the Kdm7a depletion ESCs, H3K27me2 and H3K9me2 are enriched at the transcription start site of FGF4 gene, where there are KDM7A binding regions, indicating that KDM7A is a dual-specific histone demethylase (Huang et al. 2010b). In early chick embryo in vivo, Kdm7a is preferentially expressed in the epiblast cells.

Intrinsic regulations in neural fate commitment

Consistently, overexpression of Kdm7a in chick embryos results in enlarging neural tissue, whereas the formation of the neural plate is compromised with the Kdm7a depletion (Huang et al. 2010a). Inhibition of KDM7 orthologs in zebrafish causes defects in developing brain (Tsukada et al. 2010). Clearly, the expression and function of Kdm7a is evolutionarily conserved in the early neural development. DNA methylation/demethylation DNA methylation is crucial for genomic imprinting, Xchromosome inactivation, gene expression regulation, as well as epigenetic modulation. DNA methylation profile is dynamically remodeled during the development of mammalian nervous system (Lister et al. 2013). DNA methylation is catalyzed by DNA methyltransferase 1 (DNMT1), DNMT3A and DNMT3B at cytosine residues in CpG dinucleotides (Li 2002). DNA methylation is usually related to the gene repression, which is achieved through blocking the binding of transcription factors onto DNA (Marin-Husstege et al. 2002; Hockly et al. 2003; Hao et al. 2004; Hsieh et al. 2004). In mouse, the expression of Dnmt3a was detected in embryonic ectoderm and with low levels in mesodermal cells at E7.5, and was examined ubiquitously in somites and the ventral part of the embryo at E8.5 and E9.5. The expression of Dnmt3b is high expressed in the embryonic ectoderm, neural ectoderm, and chorionic ectoderm at E7.5, and is readily detected in the forebrain and the eye at E8.5 and E9.5 (Okano et al. 1999). However, the early lethality of Dnmt1 / or Dnmt3a / ;Dnmt3b / null mice prevents the investigation of the mechanism that determines how DNA methyltransferases participate in the regulation of the neural fate commitment (Li et al. 1992; Okano et al. 1999). DNA demethylation is associated with the gene activation (Reik 2007). The status of DNA methylation is dynamic in development (Barreto et al. 2007; Reik 2007; Rai et al. 2008). During the transition from ESCs to neural lineage, CpGs at some genomic loci, such as the neural related distal regulatory elements, are demethylated, whereas CpGs at many non-neural gene loci are methylated (Mohn et al. 2008). Ten to 11 translocation (Tet) proteins are involved in genomewide dynamic changes of DNA methylation. In mammalian, there are three Tet proteins, Tet1, Tet2, and Tet3. Tet proteins catalyze 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) through oxidation reactions (Tahiliani et al. 2009; Ito et al. 2010, 2011; He et al. 2011; Koh et al. 2011). 5hmC level is high in mESCs, is reduced dramatically at the early stages of differentiation, and is

115

increased in the fully differentiated cells (Kriaucionis & Heintz 2009; Tahiliani et al. 2009; Szwagierczak et al. 2010), which supports that the modulation of DNA methylation is also dynamic. 5hmC and Tet1 are enriched at promoters of the poised genes in ESCs in vitro (Wu et al. 2011a,b). The expression of Tet1 is detected at one cell embryo, and is enriched in the cells of inner cell mass (Ito et al. 2010). In the Tet1 deficient pluripotent mESCs, the differentiation of trophectoderm is promoted, with the enhanced expression of key trophectoderm genes Cdx2, Eomes, Hand1, and Elf5, whereas neuroectoderm is reduced, accompanying the decreased expression of neural lineage genes Pax6 and NeuroD2 (Ito et al. 2010; Ficz et al. 2011; Koh et al. 2011; Williams et al. 2011; Xu et al. 2011; Dawlaty et al. 2013). In vivo, hmCG is enriched within active genomic loci in both fetal and adult mouse brain (Lister et al. 2013). Tet1 participates in the epigenetic regulation of the proliferation of adult neural stem cells (NSCs) (Zhang et al. 2013), as well as the modulation of synaptic plasticity and memory extinction (Rudenko et al. 2013). The generation of NPCs in the Tet1 / null background and the early formation of three germ layers in Tet1 / ;Tet2 / mice seems normal, probably because of the functional compensation among three Tet genes (Dawlaty et al. 2011, 2013). Loss-of-function of Tet3 gene in mouse blocks paternal gene activation, and results in early developmental failure (Gu et al. 2011). Nevertheless, so far, the function of Tet protein activity in the early neural fate specification in vivo has not been fully elucidated. Histone acetylation/deacetylation Histone lysine acetylation probably is the best-characterized histone modification, and is involved in two groups of enzymes, histone acetyltransferases, HATs, and histone deacetylases, HDACs. Acetylation of histone mediated by HATs alleviates the interaction between histone and DNA, and transcription factors can access to the DNA to activate gene expression. Deacetylation catalyzed by HDACs enhances the association between histone and DNA, and prevents the binding of transcription factors, which leads to gene repression (Jenuwein & Allis 2001). CBP and p300 acetylate histone, as well as non-histone proteins. Both CBP and p300 gene null mutant mice die at early embryonic stages with defects in the central nervous system (CNS) (Yao et al. 1998; Tanaka et al. 2000). In mammals, HDACs are generally divided into four classes: class 1 HDACs (HDAC1-3 and HDAC8), class 2 (HDAC4-7, HDAC9 and HDAC10), class 3 (sirtuins), and class 4 (HDAC11). HDAC1 and HDAC2 are the most-studied HDACs. In mouse, the nucleus expresª 2015 Japanese Society of Developmental Biologists

116

K. Tang et al.

sion of HDAC1 protein is detected at the 4-cell, 8-cell, morula and blastocyst stages, and the expression of HDAC1 transcripts is high in the head fold and neural fold at E8.5. HDAC1-null mutant mice die before E10.5 with severe defects in the CNS (Lagger et al. 2002). Both HDAC1 and HDAC2 participate in the regulation of the oligodendrocyte differentiation and the synapse formation (Akhtar et al. 2009; Ye et al. 2009; Chen et al. 2011; Jacob et al. 2011). One of the latest studies reveals that inhibition of histone deacetylation promotes the differentiation of human pluripotent stem cells (hPSCs) to NPCs. HDAC inhibitor (HDACi) treatment gives rise to more NPCs by enhancing the expression of neuroectoderm markers, as well as the neuroectoderm specification, and class I HDACs play essential roles in the NPC generation. Assays with shRNAs mediated gene knockdown approach further demonstrate that HDAC3 but not HDAC1 or HDAC2 participates in NPC differentiation through forming regulatory complex with SMRT (Yang et al. 2014). However, the contribution of histone acetylation/deacetylation to the initiation of the neural tissue has not been clearly addressed.

Perspectives The neural fate commitment is a complicated, dynamic and sequential event, which is controlled spatiotemporally. The initiation of neural plate is determined by both the extracellular signals such as BMPs, FGFs, NODAL, and WNT and the intracellular molecules including both transcriptional and epigenetic factors. The extrinsic regulation of the neural fate determination has been well studied for decades, and the intrinsic modulation, especially the epigenetic modifications of both histone and DNA, on the neural induction is one of the hottest topics in the neuroscience field. The detailed molecular mechanisms, how the extrinsic regulation and intrinsic modulation orchestrate with each other to program the neural fate commitment has just started to emerge. The advance of single cell sequencing and bioinformatics could provide instant and accurate information of not only gene expression profile, but also the instructive interaction between the extracellular and the intracellular networks. The development of micro chromatin immunoprecipitation (micro-ChIP), ChIP-seq, DNA modification assays and other techniques generates novel evidence about the gene regulation at different levels, such as the recruitment of transcription factors and epigenetic factors, histone protein modifications, and DNA methylation. All of these newly developing approaches, together with laser capture micro-dissection technique, make the analysis of the gene expression and the gene regulation at the single ª 2015 Japanese Society of Developmental Biologists

cell resolution in the tiny early embryo in situ available, which will provide accurate regulatory atlas to uncover the secrets of the neural fate commitment. The initiation of the neural tissue is achieved by the cooperation of multiple genes, extracellular signals, intracellular factors, and epigenetic molecules. In the past decades, a great amount of mouse animal models have been generated to investigate the gene functions in vivo. However, the possible compensations among a group of genes in the same subfamily, or among several extrinsic and intrinsic networks with similar functions prevent better understanding of the naive regulatory mechanism in the cell fate determination. It takes an extremely long time and great efforts to obtain a double or triple gene knockout mouse model through the traditional process. Since the first report in 2012 (Jinek et al. 2012), the CRISPR/Cas9 system has been successfully used to generate the depletion of multiple genes in both ESCs in vitro and mouse in vivo in a single treatment (Wang et al. 2013), which demonstrates a simple, fast and efficient approach to create mouse models with the genome editing on multiple genes. Moreover, the CRISPR/Cas9 system breaks the limitation of the usage of embryonic stem cells, indicating that the precise gene editing probably could be achieved on any organism in the animal kingdom. Indeed, the first twin cynomolgus monkeys with customized mutations were generated with the Cas9 system (Niu et al. 2014). CRISPR/Cas9 mediated genome editing of multiple neural genes in one mouse model and the creation of a primate model will broaden our knowledge on the neural fate commitment, which will further benefit the prevention and treatment of neural development related diseases, such as autism spectrum disorders, schizophrenia, and depression.

References Akhtar, M. W., Raingo, J., Nelson, E. D., Montgomery, R. L., Olson, E. N., Kavalali, E. T. & Monteggia, L. M. 2009. Histone deacetylases 1 and 2 form a developmental switch that controls excitatory synapse maturation and function. J. Neurosci. 29, 8288–8297. Arman, E., Haffner-Krausz, R., Chen, Y., Heath, J. K. & Lonai, P. 1998. Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. Proc. Natl Acad. Sci. USA 95, 5082–5087. Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian, N. & Lovell-Badge, R. 2003. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140. Azuara, V., Perry, P., Sauer, S., Spivakov, M., Jorgensen, H. F., John, R. M., Gouti, M., Casanova, M., Warnes, G., Merkenschlager, M. & Fisher, A. G. 2006. Chromatin signatures of pluripotent cell lines. Nat. Cell Biol. 8, 532–538. Barreto, G., Schafer, A., Marhold, J., Stach, D., Swaminathan, S. K., Handa, V., Doderlein, G., Maltry, N., Wu, W., Lyko, F. &

Intrinsic regulations in neural fate commitment

Niehrs, C. 2007. Gadd45a promotes epigenetic gene activation by repair-mediated DNA demethylation. Nature 445, 671–675. Bernstein, B. E., Mikkelsen, T. S., Xie, X. H., Kamal, M., Huebert, D. J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, K., Jaenisch, R., Wagschal, A., Feil, R., Schreiber, S. L. & Lander, E. S. 2006. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326. Blum, M., Gaunt, S. J., Cho, K. W. Y., Steinbeisser, H., Blumberg, B., Bittner, D. & Derobertis, E. M. 1992. Gastrulation in the mouse – the role of the homeobox gene goosecoid. Cell 69, 1097–1106. Bond, H. M., Mesuraca, M., Amodio, N., Mega, T., Agosti, V., Fanello, D., Pelaggi, D., Bullinger, L., Grieco, M., Moore, M. A. S., Venuta, S. & Morrone, G. 2008. Early hematopoietic zinc finger protein-zinc finger protein 521: a candidate regulator of diverse immature cells. Int. J. Biochem. Cell Biol. 40, 848–854. Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. R., Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R. G., Gifford, D. K., Melton, D. A., Jaenisch, R. & Young, R. A. 2005. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956. Boyer, L. A., Plath, K., Zeitlinger, J., Brambrink, T., Medeiros, L. A., Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M. K., Bell, G. W., Otte, A. P., Vidal, M., Gifford, D. K., Young, R. A. & Jaenisch, R. 2006. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349–353. Bracken, A. P., Dietrich, N., Pasini, D., Hansen, K. H. & Helin, K. 2006. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 20, 1123–1136. Brons, I. G., Smithers, L. E., Trotter, M. W., Rugg-Gunn, P., Sun, B., de Sousa Chuva Lopes, S. M., Howlett, S. K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R. A. & Vallier, L. 2007. Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature 448, 191–195. Burgold, T., Spreafico, F., De Santa, F., Totaro, M. G., Prosperini, E., Natoli, G. & Testa, G. 2008. The histone H3 lysine 27specific demethylase Jmjd3 Is required for neural commitment. PLoS One 3, e3034. Bylund, M., Andersson, E., Novitch, B. G. & Muhr, J. 2003. Vertebrate neurogenesis is counteracted by So1–3 activity. Nat. Neurosci. 6, 1162–1168. Cao, R. & Zhang, Y. 2004. The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3. Curr. Opin. Genet. Dev. 14, 155–164. Chen, X., Xu, H., Yuan, P., Fang, F., Huss, M., Vega, V. B., Wong, E., Orlov, Y. L., Zhang, W. W., Jiang, J. M., Loh, Y. H., Yeo, H. C., Yeo, Z. X., Narang, V., Govindarajan, K. R., Leong, B., Shahab, A., Ruan, Y. J., Bourque, G., Sung, W. K., Clarke, N. D., Wei, C. L. & Ng, H. H. 2008. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117. Chen, Y., Wang, H. B., Yoon, S. O., Xu, X. M., Hottiger, M. O., Svaren, J., Nave, K. A., Kim, H. A., Olson, E. N. & Lu, Q. R. 2011. HDAC-mediated deacetylation of NF-kappa B is critical for Schwann cell myelination. Nat. Neurosci. 14, 437–U459. Chng, Z. Z., Teo, A., Pedersen, R. A. & Vallier, L. 2010. SIP1 mediates cell-fate decisions between neuroectoderm and mesendoderm in human pluripotent stem cells. Cell Stem Cell 6, 59–70. Comijn, J., Berx, G., Vermassen, P., Verschueren, K., van Grunsven, L., Bruyneel, E., Mareel, M., Huylebroeck, D. & van Roy, F. 2001. The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol. Cell 7, 1267–1278. Coskun, V., Tsoa, R. & Sun, Y. E. 2012. Epigenetic regulation of stem cells differentiating along the neural lineage. Curr. Opin. Neurobiol. 22, 762–767.

117

Dahl, J. A., Taranger, C. K. & Collas, P. 2007. Epigenetic dynamics of pluripotency genes in the context of differentiation and nuclear reprogramming determined by Q2ChIP, a quick and quantitative chromatin immunoprecipitation technique applicable to small cell samples. Reprod. Fert. Develop. 19, 227–228. Dawlaty, M. M., Ganz, K., Powell, B. E., Hu, Y. C., Markoulaki, S., Cheng, A. W., Gao, Q., Kim, J., Choi, S. W., Page, D. C. & Jaenisch, R. 2011. Tet1 Is dispensable for maintaining pluripotency and its loss is compatible with embryonic and postnatal development. Cell Stem Cell 9, 166–175. Dawlaty, M. M., Breiling, A., Le, T., Raddatz, G., Barrasa, M. I., Cheng, A. W., Gao, Q., Powell, B. E., Li, Z., Xu, M. J., Faull, K. F., Lyko, F. & Jaenisch, R. 2013. Combined deficiency of Tet1 and Tet2 causes epigenetic abnormalities but is compatible with postnatal development. Dev. Cell 24, 310–323. De Robertis, E. M. & Kuroda, H. 2004. Dorsal-ventral patterning and neural induction in Xenopus embryos. Annu. Rev. Cell Dev. Biol. 20, 285–308. Dey, B. K., Stalker, L., Schnerch, A., Bhatia, M., Taylor-Papidimitriou, J. & Wynder, C. 2008. The histone demethylase KDM5b/JARID1b plays a role in cell fate decisions by blocking terminal differentiation. Mol. Cell. Biol. 28, 5312–5327. Di-Gregorio, A., Sancho, M., Stuckey, D. W., Crompton, L. A., Godwin, J., Mishina, Y. & Rodriguez, T. A. 2007. BMP signalling inhibits premature neural differentiation in the mouse embryo. Development 134, 3359–3369. Feldman, B., Poueymirou, W., Papaioannou, V. E., Dechiara, T. M. & Goldfarb, M. 1995. Requirement of FGF-4 for postimplantation mouse development. Science 267, 246–249. Ficz, G., Branco, M. R., Seisenberger, S., Santos, F., Krueger, F., Hore, T. A., Marques, C. J., Andrews, S. & Reik, W. 2011. Dynamic regulation of 5-hydroxymethylcytosine in mouse ES cells and during differentiation. Nature 473, 398–U589. Finley, M. F., Devata, S. & Huettner, J. E. 1999. BMP-4 inhibits neural differentiation of murine embryonic stem cells. J. Neurobiol. 40, 271–287. Frankenberg, S., Smith, L., Greenfield, A. & Zernicka-Goetz, M. 2007. Novel gene expression patterns along the proximodistal axis of the mouse embryo before gastrulation. BMC Dev. Biol. 7, 8. Glaser, T. & Brustle, O. 2005. Retinoic acid induction of ES-cellderived neurons: the radial glia connection. Trends Neurosci. 28, 397–400. Golebiewska, A., Atkinson, S. P., Lako, M. & Armstrong, L. 2009. Epigenetic landscaping during hESC differentiation to neural cells. Stem Cells 27, 1298–1308. Graham, V., Khudyakov, J., Ellis, P. & Pevny, L. 2003. SOX2 functions to maintain neural progenitor identity. Neuron 39, 749–765. Gu, T. P., Guo, F., Yang, H., Wu, H. P., Xu, G. F., Liu, W., Xie, Z. G., Shi, L. Y., He, X. Y., Jin, S. G., Iqbal, K., Shi, Y. J. G., Deng, Z. X., Szabo, P. E., Pfeifer, G. P., Li, J. S. & Xu, G. L. 2011. The role of Tet3 DNA dioxygenase in epigenetic reprogramming by oocytes. Nature 477, 606–610. Gubbay, J., Collignon, J., Koopman, P., Capel, B., Economou, A., Munsterberg, A., Vivian, N., Goodfellow, P. & Lovellbadge, R. 1990. A gene-mapping to the sex-determining region of the mouse Y-chromosome is a member of a novel family of embryonically expressed genes. Nature 346, 245– 250. Hao, Y. L., Creson, T., Zhang, L., Li, P. P., Du, F., Yuan, P. X., Gould, T. D., Manji, H. K. & Chen, G. 2004. Mood stabilizer valproate promotes ERK pathway-dependent cortical neuronal growth and neurogenesis. J. Neurosci. 24, 6590–6599. Hatta, K. & Takahashi, Y. 1996. Secondary axis induction by heterospecific organizers in zebrafish. Dev. Dyn. 205, 183–195. He, Y. F., Li, B. Z., Li, Z., Liu, P., Wang, Y., Tang, Q. Y., Ding, J. P., Jia, Y. Y., Chen, Z. C., Li, L., Sun, Y., Li, X. X., Dai, Q., Song, C. X., Zhang, K. L., He, C. & Xu, G. L. 2011. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307.

ª 2015 Japanese Society of Developmental Biologists

118

K. Tang et al.

Hemmatibrivanlou, A. & Melton, D. 1997. Vertebrate embryonic cells will become nerve cells unless told otherwise. Cell 88, 13–17. Hirabayashi, Y. & Gotoh, Y. 2010. Epigenetic control of neural precursor cell fate during development. Nat. Rev. Neurosci. 11, 377–388. Hockly, E., Richon, V. M., Woodman, B., Smith, D. L., Zhou, X. B., Rosa, E., Sathasivam, K., Ghazi-Noori, S., Mahal, A., Lowden, P. A. S., Steffan, J. S., Marsh, J. L., Thompson, L. M., Lewis, C. M., Marks, P. A. & Bates, G. P. 2003. Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington’s disease. Proc. Natl Acad. Sci. USA 100, 2041–2046. Hsieh, J. & Gage, F. H. 2005. Chromatin remodeling in neural development and plasticity. Curr. Opin. Cell Biol. 17, 664– 671. Hsieh, J., Nakashima, K., Kuwabara, T., Mejia, E. & Gage, F. H. 2004. Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc. Natl Acad. Sci. USA 101, 16659–16664. Huang, C. Y., Chen, J., Zhang, T., Zhu, Q. Q., Xiang, Y., Chen, C. D. & Jing, N. H. 2010a. The dual histone demethylase KDM7A promotes neural induction in early chick embryos. Dev. Dyn. 239, 3350–3357. Huang, C. Y., Xiang, Y., Wang, Y. R., Li, X., Xu, L. Y., Zhu, Z. Q., Zhang, T., Zhu, Q. Q., Zhang, K. J., Jing, N. H. & Chen, C. D. 2010b. Dual-specificity histone demethylase KIAA1718 (KDM7A) regulates neural differentiation through FGF4. Cell Res. 20, 154–165. Ito, S., D’alessio, A. C., Taranova, O. V., Hong, K., Sowers, L. C. & Zhang, Y. 2010. Role of Tet proteins in 5mC to 5hmC conversion, ES-cell self-renewal and inner cell mass specification. Nature 466, 1129–U1151. Ito, S., Shen, L., Dai, Q., Wu, S. C., Collins, L. B., Swenberg, J. A., He, C. & Zhang, Y. 2011. Tet proteins can convert 5methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science 333, 1300–1303. Iwafuchi-Doi, M., Yoshida, Y., Onichtchouk, D., Leichsenring, M., Driever, W., Takemoto, T., Uchikawa, M., Kamachi, Y. & Kondoh, H. 2011. The Pou5f1/Pou3f-dependent but SoxBindependent regulation of conserved enhancer N2 initiates Sox2 expression during epiblast to neural plate stages in vertebrates. Dev. Biol. 352, 354–366. Iwafuchi-Doi, M., Matsuda, K., Murakami, K., Niwa, H., Tesar, P. J., Aruga, J., Matsuo, I. & Kondoh, H. 2012. Transcriptional regulatory networks in epiblast cells and during anterior neural plate development as modeled in epiblast stem cells. Development 139, 3926–3937. Jacob, C., Christen, C. N., Pereira, J. A., Somandin, C., Baggiolini, A., Lotscher, P., Ozcelik, M., Tricaud, N., Meijer, D., Yamaguchi, T., Matthias, P. & Suter, U. 2011. HDAC1 and HDAC2 control the transcriptional program of myelination and the survival of Schwann cells. Nat. Neurosci. 14, 429– U453. Jenuwein, T. & Allis, C. D. 2001. Translating the histone code. Science 293, 1074–1080. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A. & Charpentier, E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821. Kamiya, D., Banno, S., Sasai, N., Ohgushi, M., Inomata, H., Watanabe, K., Kawada, M., Yakura, R., Kiyonari, H., Nakao, K., Jakt, L. M., Nishikawa, S. & Sasai, Y. 2011. Intrinsic transition of embryonic stem-cell differentiation into neural progenitors. Nature 470, 503–U592. Kim, J. W., Chu, J. L., Shen, X. H., Wang, J. L. & Orkin, S. H. 2008. An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132, 1049–1061. Kintner, C. R. & Dodd, J. 1991. Hensen node induces neural tissue in Xenopus ectoderm – implications for the action of the organizer in neural induction. Development 113, 1495–1505.

ª 2015 Japanese Society of Developmental Biologists

Koh, K. P., Yabuuchi, A., Rao, S., Huang, Y., Cunniff, K., Nardone, J., Laiho, A., Tahiliani, M., Sommer, C. A., Mostoslavsky, G., Lahesmaa, R., Orkin, S. H., Rodig, S. J., Daley, G. Q. & Rao, A. 2011. Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 8, 200–213. Kriaucionis, S. & Heintz, N. 2009. The nuclear DNA Base 5-hydroxymethylcytosine is present in purkinje neurons and the brain. Science 324, 929–930. Kunath, T., Saba-El-Leil, M. K., Almousailleakh, M., Wray, J., Meloche, S. & Smith, A. 2007. FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 134, 2895–2902. Lagger, G., O’Carroll, D., Rembold, M., Khier, H., Tischler, J., Weitzer, G., Schuettengruber, B., Hauser, C., Brunmeir, R., Jenuwein, T. & Seiser, C. 2002. Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J. 21, 2672–2681. Lee, T. I., Jenner, R. G., Boyer, L. A., Guenther, M. G., Levine, S. S., Kumar, R. M., Chevalier, B., Johnstone, S. E., Cole, M. F., Isono, K., Koseki, H., Fuchikami, T., Abe, K., Murray, H. L., Zucker, J. P., Yuan, B. B., Bell, G. W., Herbolsheimer, E., Hannett, N. M., Sun, K. M., Odom, D. T., Otte, A. P., Volkert, T. L., Bartel, D. P., Melton, D. A., Gifford, D. K., Jaenisch, R. & Young, R. A. 2006. Control of developmental regulator’s by polycomb in human embryonic stem cells. Cell 125, 301–313. Lee, E. R., Murdoch, F. E. & Fritsch, M. K. 2007. High histone acetylation and decreased polycomb repressive complex 2 member levels regulate gene specific transcriptional changes during early embryonic stem cell differentiation induced by retinoic acid. Stem Cells 25, 2191–2199. Lerchner, W., Latinkic, B. V., Remacle, J. E., Huylebroeck, D. & Smith, J. C. 2000. Region-specific activation of the Xenopus Brachyury promoter involves active repression in ectoderm and endoderm: a study using transgenic frog embryos. Development 127, 2729–2739. Li, E. 2002. Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3, 662–673. Li, E., Bestor, T. H. & Jaenisch, R. 1992. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926. Lister, R., Mukamel, E. A., Nery, J. R., Urich, M., Puddifoot, C. A., Johnson, N. D., Lucero, J., Huang, Y., Dwork, A. J., Schultz, M. D., Yu, M., Tonti-Filippini, J., Heyn, H., Hu, S. J., Wu, J. C., Rao, A., Esteller, M., He, C., Haghighi, F. G., Sejnowski, T. J., Behrens, M. M. & Ecker, J. R. 2013. Global epigenomic reconfiguration during mammalian brain development. Science 341, 1237905. Marin-Husstege, M., Muggironi, M., Liu, A. X. & Casaccia-Bonnefil, P. 2002. Histone deacetylase activity is necessary for oligodendrocyte lineage progression. J. Neurosci. 22, 10333–10345. Masui, S., Nakatake, Y., Toyooka, Y., Shimosato, D., Yagi, R., Takahashi, K., Okochi, H., Okuda, A., Matoba, R., Sharov, A. A., Ko, M. S. H. & Niwa, H. 2007. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat. Cell Biol. 9, 625–U626. Meijer, D., Graus, A., Kraay, R., Langeveld, A., Mulder, M. P. & Grosveld, G. 1990. The octamer binding-factor Oct6 – cDNA cloning and expression in early embryonic-cells. Nucleic Acids Res. 18, 7357–7365. Meyers, E. N., Lewandoski, M. & Martin, G. R. 1998. An Fgf8 mutant allelic series generated by Cre- and Flp-mediated recombination. Nat. Genet. 18, 136–141. Mikkelsen, T. S., Ku, M. C., Jaffe, D. B., Issac, B., Lieberman, E., Giannoukos, G., Alvarez, P., Brockman, W., Kim, T. K., Koche, R. P., Lee, W., Mendenhall, E., O’Donovan, A., Presser, A., Russ, C., Xie, X. H., Meissner, A., Wernig, M., Jaenisch, R., Nusbaum, C., Lander, E. S. & Bernstein, B. E.

Intrinsic regulations in neural fate commitment

2007. Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448, 553–560. Mishina, Y., Suzuki, A., Ueno, N. & Behringer, R. R. 1995. Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 9, 3027–3037. Mohn, F., Weber, M., Rebhan, M., Roloff, T. C., Richter, J., Stadler, M. B., Bibel, M. & Schubeler, D. 2008. Lineage-specific polycomb targets and de novo DNA methylation define restriction and potential of neuronal progenitors. Mol. Cell 30, 755–766. Monuki, E. S., Weinmaster, G., Kuhn, R. & Lemke, G. 1989. SCIP – a glial POU domain gene regulated by cyclic-AMP. Neuron 3, 783–793. Munoz-Sanjuan, I. & Brivanlou, A. H. 2002. Neural induction, the default model and embryonic stem cells. Nat. Rev. Neurosci. 3, 271–280. Niu, Y. Y., Shen, B., Cui, Y. Q., Chen, Y. C., Wang, J. Y., Wang, L., Kang, Y., Zhao, X. Y., Si, W., Li, W., Xiang, A. P., Zhou, J. K., Guo, X. J., Bi, Y., Si, C. Y., Hu, B., Dong, G. Y., Wang, H., Zhou, Z. M., Li, T. Q., Tan, T., Pu, X. Q., Wang, F., Ji, S. H., Zhou, Q., Huang, X. X., Ji, W. Z. & Sha, J. H. 2014. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156, 836–843. Okano, M., Bell, D. W., Haber, D. A. & Li, E. 1999. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257. Oppenheimer, J. M. 1936a. Structures developed in amphibians by implantation of living fish organizer. Exp. Biol. Med. 34, 461–463. Oppenheimer, J. M. 1936b. Transplantation experiments on developing teleosts (Fundulus and Perca). J. Exp. Zool. 72, 409–437. Orkin, S. H. & Hochedlinger, K. 2011. Chromatin connections to pluripotency and cellular reprogramming. Cell 145, 835–850. Outchkourov, N. S., Muino, J. M., Kaufmann, K., van Ijcken, W. F. J., Koerkamp, M. J. G., van Leenen, D., de Graaf, P., Holstege, F. C. P., Grosveld, F. G. & Timmers, H. T. M. 2013. Balancing of histone H3K4 methylation states by the Kdm5c/SMCX histone demethylase modulates promoter and enhancer function. Cell Rep. 3, 1071–1079. Pasini, D., Bracken, A. P., Hansen, J. B., Capillo, M. & Helin, K. 2007. The polycomb group protein Suz12 is required for embryonic stem cell differentiation. Mol. Cell. Biol. 27, 3769– 3779. Postigo, A. A., Depp, J. L., Taylor, J. J. & Kroll, K. L. 2003. Regulation of Smad signaling through a differential recruitment of coactivators and corepressors by ZEB proteins. EMBO J. 22, 2453–2462. Rai, K., Huggins, I. J., James, S. R., Karpf, A. R., Jones, D. A. & Cairns, B. R. 2008. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45. Cell 135, 1201–1212. Reik, W. 2007. Stability and flexibility of epigenetic gene regulation in mammalian development. Nature 447, 425–432. Rex, M., Uwanogho, D. A., Orme, A., Scotting, P. J. & Sharpe, P. T. 1997. cSox21 exhibits a complex and dynamic pattern of transcription during embryonic development of the chick central nervous system. Mech. Dev. 66, 39–53. Roidl, D. & Hacker, C. 2014. Histone methylation during neural development. Cell Tissue Res. 356, 539–552. Ross, S. A., Mccaffery, P. J., Drager, U. C. & De Luca, L. M. 2000. Retinoids in embryonal development. Physiol. Rev. 80, 1021–1054. Rudenko, A., Dawlaty, M. M., Seo, J., Cheng, A. W., Meng, J., Le, T., Faull, K. F., Jaenisch, R. & Tsai, L. H. 2013. Tet1 is critical for neuronal activity-regulated gene expression and memory extinction. Neuron 79, 1109–1122. Sasai, Y. & Derobertis, E. M. 1997. Ectodermal patterning in vertebrate embryos. Dev. Biol. 182, 5–20.

119

Schmitz, S. U., Albert, M., Malatesta, M., Morey, L., Johansen, J. V., Bak, M., Tommerup, N., Abarrategui, I. & Helin, K. 2011. Jarid1b targets genes regulating development and is involved in neural differentiation. EMBO J. 30, 4586–4600. Scholer, H. R., Hatzopoulos, A. K., Balling, R., Suzuki, N. & Gruss, P. 1989. A family of octamer-specific proteins present during mouse embryogenesis – evidence for germline-specific expression of an Oct factor. EMBO J. 8, 2543–2550. Sheng, G. J., Dos Reis, M. & Stern, C. D. 2003. Churchill, a zinc finger transcriptional activator, regulates the transition between gastrulation and neurulation. Cell 115, 603–613. Sher, F., Rossler, R., Brouwer, N., Balasubramaniyan, V., Boddeke, E. & Copray, S. 2008. Differentiation of neural stem cells into oligodendrocytes: involvement of the polycomb group protein Ezh2. Stem Cells 26, 2875–2883. Sims, R. J., Nishioka, K. & Reinberg, D. 2003. Histone lysine methylation: a signature for chromatin function. Trends Genet. 19, 629–639. Spemann, H. 1921. The production of animal chimaera by heteroplastic embryonal transplantation between triton cristatus and taeniatus. Arch. Entw. Org. 48, 533–570. Spemann, H. & Mangold, H. 1924. The induction of embryonic predispositions by implantation of organizers foreign to the species. Arch. Mikrosk. Anat. 100, 599–638. Stavridis, M. P., Lunn, J. S., Collins, B. J. & Storey, K. G. 2007. A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. Development 134, 2889–2894. Stern, C. D. 2005. Neural induction: old problem, new findings, yet more questions. Development 132, 2007–2021. Stern, C. D. 2006. Neural induction: 10 years on since the ‘default model’. Curr. Opin. Cell Biol. 18, 692–697. Sterneckert, J., Stehling, M., Bernemann, C., Arauzo-Bravo, M. J., Greber, B., Gentile, L., Ortmeier, C., Sinn, M., Wu, G., Ruau, D., Zenke, M., Brintrup, R., Klein, D. C., Ko, K. & Scholer, H. R. 2010. Neural induction intermediates exhibit distinct roles of Fgf signaling. Stem Cells 28, 1772–1781. Sun, X., Meyers, E. N., Lewandoski, M. & Martin, G. R. 1999. Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 13, 1834–1846. Suzuki, N., Rohdewohld, H., Neuman, T., Gruss, P. & Scholer, H. R. 1990. Oct-6 – a POU transcription factor expressed in embryonal stem-cells and in the developing brain. EMBO J. 9, 3723–3732. Szutorisz, H., Canzonetta, C., Georgiou, A., Chow, C. M., Tora, L. & Dillon, N. 2005. Formation of an active tissue-specific chromatin domain initiated by epigenetic marking at the embryonic stem cell stage. Mol. Cell. Biol. 25, 1804–1820. Szwagierczak, A., Bultmann, S., Schmidt, C. S., Spada, F. & Leonhardt, H. 2010. Sensitive enzymatic quantification of 5hydroxymethylcytosine in genomic DNA. Nucleic Acids Res. 38, e181. Tahiliani, M., Koh, K. P., Shen, Y. H., Pastor, W. A., Bandukwala, H., Brudno, Y., Agarwal, S., Iyer, L. M., Liu, D. R., Aravind, L. & Rao, A. 2009. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 324, 930–935. Takemoto, T., Uchikawa, M., Yoshida, M., Bell, D. M., LovellBadge, R., Papaioannou, V. E. & Kondoh, H. 2011. Tbx6dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells. Nature 470, 394–398. Tanaka, Y., Naruse, I., Hongo, T., Xu, M. J., Nakahata, T., Maekawa, T. & Ishii, S. 2000. Extensive brain hemorrhage and embryonic lethality in a mouse null mutant of CREB-binding protein. Mech. Dev. 95, 133–145. Tanaka, S., Kamachi, Y., Tanouchi, A., Hamada, H., Jing, N. H. & Kondoh, H. 2004. Interplay of SOX and POU factors in regulation of the Nestin gene in neural primordial cells. Mol. Cell. Biol. 24, 8834–8846. Tesar, P. J., Chenoweth, J. G., Brook, F. A., Davies, T. J., Evans, E. P., Mack, D. L., Gardner, R. L. & McKay, R. D.

ª 2015 Japanese Society of Developmental Biologists

120

K. Tang et al.

2007. New cell lines from mouse epiblast share defining features with human embryonic stem cells. Nature 448, 196– 199. Thomson, M., Liu, S. J., Zou, L. N., Smith, Z., Meissner, A. & Ramanathan, S. 2011. Pluripotency factors in embryonic stem cells regulate differentiation into germ layers. Cell 145, 875–889. Tropepe, V., Hitoshi, S., Sirard, C., Mak, T. W., Rossant, J. & Van Der Kooy, D. 2001. Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30, 65–78. Tsukada, Y. I., Ishitani, T. & Nakayama, K. I. 2010. KDM7 is a dual demethylase for histone H3 Lys 9 and Lys 27 and functions in brain development. Genes Dev. 24, 432–437. Uchikawa, M., Ishida, Y., Takemoto, T., Kamachi, Y. & Kondoh, H. 2003. Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev. Cell 4, 509–519. Verschueren, K., Remacle, J. E., Collart, C., Kraft, H., Baker, B. S., Tylzanowski, P., Nelles, L., Wuytens, G., Su, M. T., Bodmer, R., Smith, J. C. & Huylebroeck, D. 1999. SIP1, a novel zinc finger homeodomain repressor, interacts with Smad proteins and binds to 5 ‘-CACCT sequences in candidate target genes. J. Biol. Chem. 274, 20489–20498. Vonica, A. & Brivanlou, A. H. 2006. An obligatory caravanserai stop on the silk road to neural induction: inhibition of BMP/ GDF signaling. Semin. Cell Dev. Biol. 17, 117–132. Waddington, C. H. 1932. Experiments on the development of chick and duck embryos, cultivated in vitro. Philos. Trans. R. Soc. Lond. B Biol. Sci. 221, 179–230. Waddington, C. H. 1933. Induction by the primitive streak and its derivatives in the chick. J. Exp. Biol. 10, 38–46. Waddington, C. H. 1934. Experiments on embryonic induction Part I. The competence of the extra-embryonic ectoderm in the chick Part II. Experiments on coagulated organisers in the chick Part III. A note on inductions by chick primitive streak transplanted to the rabbit embryo. J. Exp. Biol. 11, 211–227. Waddington, C. H. 1936. Organizers in mammalian development. Nature 138, 125–125. Wang, Z., Oron, E., Nelson, B., Razis, S. & Ivanova, N. 2012. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10, 440–454. Wang, H. Y., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F. & Jaenisch, R. 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/ Cas-mediated genome engineering. Cell 153, 910–918. Warming, S., Liu, P., Suzuki, T., Akagi, K., Lindtner, S., Pavlakis, G. N., Jenkins, N. A. & Copeland, N. G. 2003. Evi3, a common retroviral integration site in murine B-cell lymphoma, encodes an EBFAZ-related Kruppel-like zinc finger protein. Blood 101, 1934–1940. Williams, K., Christensen, J., Pedersen, M. T., Johansen, J. V., Cloos, P. A. C., Rappsilber, J. & Helin, K. 2011. TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity. Nature 473, 343–U472. Wilson, S. I. & Edlund, T. 2001. Neural induction: toward a unifying mechanism. Nat. Neurosci. 4, 1161–1168. Wissmann, M., Yin, N., Muller, J. M., Greschik, H., Fodor, B. D., Jenuwein, T., Vogler, C., Schneider, R., Gunther, T., Buettner, R., Metzger, E. & Schule, R. 2007. Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression. Nat. Cell Biol. 9, 347–353.

ª 2015 Japanese Society of Developmental Biologists

Wood, H. B. & Episkopou, V. 1999. Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages. Mech. Dev. 86, 197–201. Wu, H., D’Alessio, A. C., Ito, S., Wang, Z. B., Cui, K. R., Zhao, K. J., Sun, Y. E. & Zhang, Y. 2011a. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 25, 679–684. Wu, H., D’Alessio, A. C., Ito, S., Xia, K., Wang, Z. B., Cui, K. R., Zhao, K. J., Sun, Y. E. & Zhang, Y. 2011b. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature 473, 389–U578. Xi, H., Treacy, M. N., Simmons, D. M., Ingraham, H. A., Swanson, L. W. & Rosenfeld, M. G. 1989. Expression of a large family of POU-domain regulatory genes in mammalian brain-development. Nature 340, 35–42. Xu, Y. F., Wu, F. Z., Tan, L., Kong, L. C., Xiong, L. J., Deng, J., Barbera, A. J., Zheng, L. J., Zhang, H. K., Huang, S., Min, J. R., Nicholson, T., Chen, T. P., Xu, G. L., Shi, Y., Zhang, K. & Shi, Y. G. 2011. Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol. Cell 42, 451–464. Yamane, K., Tateishi, K., Klose, R. J., Fang, J., Fabrizio, L. A., Erdjument-Bromage, H., Taylor-Papadimitriou, J., Tempst, P. & Zhang, Y. 2007. PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation. Mol. Cell 25, 801–812. Yang, J., Tang, Y., Liu, H., Guo, F., Ni, J. & Le, W. 2014. Suppression of histone deacetylation promotes the differentiation of human pluripotent stem cells towards neural progenitor cells. BMC Biol. 12, 95. Yao, T. P., Oh, S. P., Fuchs, M., Zhou, N. D., Ch’ng, L. E., Newsome, D., Bronson, R. T., Li, E., Livingston, D. M. & Eckner, R. 1998. Gene dosage-dependent embryonic development and proliferation defects in mice lacking the transcriptional integrator p300. Cell 93, 361–372. Ye, F., Chen, Y., Hoang, T., Montgomery, R. L., Zhao, X. H., Bu, H., Hu, T., Taketo, M. M., van Es, J. H., Clevers, H., Hsieh, J., Bassel-Duby, R., Olson, E. N. & Lu, Q. R. 2009. HDAC1 and HDAC2 regulate oligodendrocyte differentiation by disrupting the beta-catenin-TCF interaction. Nat. Neurosci. 12, 829–U835. Zhang, Y. & Reinberg, D. 2001. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev. 15, 2343–2360. Zhang, K., Li, L., Huang, C., Shen, C., Tan, F., Xia, C., Liu, P., Rossant, J. & Jing, N. 2010. Distinct functions of BMP4 during different stages of mouse ES cell neural commitment. Development, 137, 2095–2105. Zhang, R. R., Cui, Q. Y., Murai, K., Lim, Y. C., Smith, Z. D., Jin, S. N., Ye, P., Rosa, L., Lee, Y. K., Wu, H. P., Liu, W., Xu, Z. M., Yang, L., Ding, Y. Q., Tang, F. C., Meissner, A., Ding, C. M., Shi, Y. H. & Xu, G. L. 2013. Tet1 regulates adult hippocampal neurogenesis and cognition. Cell Stem Cell 13, 237–245. Zhu, Q. Q., Song, L., Peng, G. D., Sun, N., Chen, J., Zhang, T., Sheng, N. Y., Tang, W., Qian, C., Qiao, Y. B., Tang, K., Han, J. D. J., Li, J. S. & Jing, N. H. 2014. The transcription factor Pou3f1 promotes neural fate commitment via. Elife 3, 02224. Zwart, R., Broos, L., Grosveld, G. & Meijer, D. 1996. The restricted expression pattern of the POU factor Oct-6 during early development of the mouse nervous system. Mech. Dev. 54, 185–194.